Quaternary ammonium salt pesticide fluorescent probe and preparation method and application thereof

Abstract

The quaternary ammonium salt pesticide fluorescent probe, a preparation method and application thereof are provided by the present disclosure, and the chemical formula of the quaternary ammonium salt pesticide fluorescent probe is C 48 H 50 N4O 10 S2, which is mainly a diphenylamine disulfonic acid derivative synthesized by using diphenylamine disulfonic acid, triethylamine and naphthalene anhydride. The probe can produce electrostatic interaction and hydrogen bond interaction with quaternary ammonium salt pesticides, and through the combined action of inner filter effect and photoinduced electron transfer, the emission light of the probe is quenched under 305nm excitation light, realizing high-sensitivity detection of quaternary ammonium salt pesticides. In combination with the fact that the self-fluorescence emission light of the diphenylamine is 340nm, and the ratio of the fluorescence intensity at 340nm and 480nm is less affected by paraquat, and the ratio has a linear relationship with the concentration of the diphenylamine, a mathematical model can be constructed to solve the concentrations of the two pesticides by using the fluorescence intensity at 340nm and 480nm, realizing the identification and quantitative detection of the diphenylamine and paraquat.

Description

Technical Field

[0001] This disclosure relates to the field of fluorescence detection technology, and in particular to a quaternary ammonium salt pesticide fluorescent probe and its preparation method and application. Background Technology

[0002] Environmental pollution is a growing concern, stemming from its direct impact on the ecological environment and human health. Therefore, monitoring and detecting trace pollutants is crucial.

[0003] Both diquat and paraquat are quaternary ammonium herbicides. As ionic pesticides, they are rapidly absorbed and metastasized in the body and are highly toxic to mammals. Due to the lack of effective treatments, poisoning by these pesticides often attracts widespread attention. After accidental ingestion of quaternary ammonium herbicides, the pesticide molecules are rapidly absorbed by the body and eventually enter tissues to exert their toxicity. Therefore, prompt diagnosis and intervention such as gastric lavage and hemoperfusion are essential, placing high demands on rapid diagnosis of poisoning.

[0004] Furthermore, while the use of paraquat is currently banned, diquat remains permitted. Due to paraquat's superior herbicidal effect, some individuals have illegally mixed it into diquat, thus creating a demand for detecting adulteration in herbicides and for detecting quaternary ammonium salt herbicides. Currently, the detection of paraquat and diquat primarily utilizes large-scale instrumental methods, employing liquid chromatography-mass spectrometry (LC-MS) to detect paraquat and diquat residues. However, this method suffers from drawbacks such as expensive equipment, the need for specialized personnel, and long processing times. Clinically, the sodium dithionite colorimetric method is used as a rapid detection method for paraquat and diquat poisoning. However, this method has limitations, including high detection limits, long processing times, and susceptibility to background interference. Moreover, once the pesticide rapidly enters tissues from the bloodstream, this method becomes undetectable. Therefore, the development of identification methods for diquat and paraquat is urgent. Furthermore, as diquat is used in agriculture as a primary herbicide and alternative to paraquat in the market, the use of diquat in agriculture also requires technicians to pay attention to pesticide residue issues. Therefore, the development of diquat pesticide residue detection methods is also necessary. Summary of the Invention

[0005] This disclosure aims to address at least one of the technical problems existing in the prior art.

[0006] Therefore, the first party to this disclosure provides a fluorescent probe for quaternary ammonium pesticides, which is a benzidine disulfonic acid derivative. It contains active groups such as carbonyl and sulfonic acid groups, enabling electrostatic and hydrogen bonding interactions with quaternary ammonium pesticides, thus achieving rapid fluorescence detection of these pesticides. The fluorescent probe for quaternary ammonium pesticides has the following structure:

[0007]

[0008] The method for preparing the above-mentioned quaternary ammonium salt pesticide fluorescent probe provided in the second aspect of this disclosure includes the following steps:

[0009] Step 1: Dissolve benzidine disulfonic acid and triethylamine in a solvent and heat under inert gas protection to obtain a homogeneous solution A;

[0010] Step 2: Add naphthalene anhydride and benzoic acid to solution A, and continue heating under the protection of the inert gas to obtain a homogeneous solution B;

[0011] Step 3: Add ethyl acetate to precipitate the solute in solution B, then wash and dry the precipitated solute with detergent to obtain the quaternary ammonium salt pesticide fluorescent probe.

[0012] In some embodiments, the solvent is m-cresol or p-cresol.

[0013] In some embodiments, the mass ratio of benzidine disulfonic acid, triethylamine and solvent is 1:1 to 5:5 to 25.

[0014] In some embodiments, the heating temperature in step one is between 60°C and 100°C, and the heating time is between 0.5h and 4h.

[0015] In some embodiments, the inert gas is selected from nitrogen, helium, or argon.

[0016] In some embodiments, the mass ratio of solution B, the naphthalene anhydride, and the benzoic acid is 1:0.2 to 1:0.3 to 1.

[0017] In some embodiments, the heating temperature in step two is between 120°C and 200°C, and the heating time is between 12h and 48h.

[0018] In some embodiments, the washing solution is selected from any one or a mixture of methanol, ethanol, ethyl acetate and acetonitrile.

[0019] The application of the quaternary ammonium salt pesticide fluorescent probe provided in the third aspect of this disclosure in the fluorescence detection of diquat concentration includes the following steps:

[0020] The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and standard solutions containing different concentrations of diquat were added to obtain multiple standard solutions. The aqueous solution of the quaternary ammonium pesticide fluorescent probe without diquat was used as the blank group, and the aqueous solution of the quaternary ammonium pesticide fluorescent probe with diquat was used as the experimental group. Each standard solution was excited with fluorescence at 305 nm, and the fluorescence spectrum of all standard solutions and the fluorescence intensity at 340 nm and 480 nm were recorded. The fluorescence intensities of the blank group at 340 nm and 480 nm were recorded as F0 and F1, respectively, and the fluorescence intensities of the experimental group at 340 nm and 480 nm were recorded as F2 and F3, respectively. The working curve for detecting the diquat concentration was obtained by fitting the relationship between y = (F2-F0) / (F3 / F1) and the diquat concentration x.

[0021] The quaternary ammonium salt pesticide fluorescent probe was dissolved in an aqueous solution, and an extract containing diquat was added to obtain a sample. The sample was subjected to fluorescence excitation at 305 nm. The fluorescence intensities F2 and F3 of the sample at 340 nm and 480 nm, and the fluorescence intensities F0 and F1 of the blank group at 340 nm and 480 nm were input into the working curve to obtain the concentration of diquat contained in the sample.

[0022] In some embodiments, the expression for the working curve is: y = 3614.1x + 4.048.

[0023] In some embodiments, the mass ratio of the quaternary ammonium salt pesticide fluorescent probe to water is 1:2000 to 200000.

[0024] The application of the aforementioned quaternary ammonium salt pesticide fluorescent probes provided in the third aspect of this disclosure in the fluorescence detection of paraquat doped with diquat includes the following steps:

[0025] Step 1: Determine the working curves for measuring the concentration of diquat and paraquat, respectively. There are two working curves for measuring the concentration of diquat, denoted as equation (1) and equation (2), respectively. The working curve for measuring the concentration of paraquat is denoted as equation (3). The determination steps are described as follows:

[0026] Determination of Equations (1) and (2): The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and aqueous solutions diluted with different concentrations of diquat technical were added to obtain multiple first standard solutions. The aqueous solution of the quaternary ammonium pesticide fluorescent probe without diquat technical dilution was used as the first blank group, and the aqueous solution of the quaternary ammonium pesticide fluorescent probe with diquat technical dilution was used as the first experimental group. Each first standard solution was excited at 305 nm, and the fluorescence spectra of all first standard solutions, as well as those at 340 nm and 480 nm, were recorded. The fluorescence intensity at 340nm and 480nm of the first blank group was recorded as F0 and F1, respectively, and the fluorescence intensity at 340nm and 480nm of the first experimental group was recorded as F2 and F3, respectively. The first working curve for detecting the concentration of diquat was obtained by fitting the relationship between y=(F2-F0) / (F3 / F1) and the concentration of diquat x, and was recorded as equation (1). At the same time, the second working curve for detecting the concentration of diquat was obtained by fitting the relationship between y=F3 / F1 and the concentration of diquat x, and was recorded as equation (2).

[0027] Determination of Equation (3): The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and different concentrations of paraquat technical diluted in aqueous solution were added to obtain a variety of second standard solutions. The quaternary ammonium pesticide fluorescent probe aqueous solution without paraquat technical diluted solution was used as the second blank group, and the quaternary ammonium pesticide fluorescent probe aqueous solution with paraquat technical diluted solution was used as the second experimental group. Each second standard solution was excited at 305 nm, and the fluorescence spectrum and fluorescence intensity at 480 nm of all second standard solutions were recorded. The fluorescence intensity at 480 nm of the second blank group was recorded as F1, and the fluorescence intensity at 480 nm of the second experimental group was recorded as F3. The working curve for detecting paraquat concentration was obtained by fitting the relationship between y = F3 / F1 and the concentration x of paraquat technical diluted solution, which is recorded as Equation (3).

[0028] Step 2, Detection of paraquat doping in diquat in the sample: Dissolve the quaternary ammonium pesticide fluorescent probe in an aqueous solution, add the diquat formulation to be tested to obtain the test solution, and use the aqueous solution of the quaternary ammonium pesticide fluorescent probe without the diquat formulation to be tested as the third blank group. Excite the test solution and the third blank group at 305 nm, and record the fluorescence intensities F2 and F3 of the test solution at 340 nm and 480 nm, and the fluorescence intensities F0 and F1 of the third blank group at 340 nm and 480 nm. Substitute y1=(F2-F0) / (F3 / F1) into equation (1) to obtain the concentration x1 of diquat in the test solution. Substitute x1 into equation (2) and record the obtained y value as y 理论 y 理论 F3 represents the fluorescence intensity of the test solution at 480 nm without paraquat doping. ’The ratio of the fluorescence intensity F1 of the test solution at 480 nm to that of the third blank group is denoted as y. 实际 If y 实际 Less than y 理论 If the test result is positive, it is determined that the diquat formulation is adulterated with paraquat, and Y = y 实际 / y 理论 Substituting into equation (3), we obtain the concentration of paraquat; if y 实际 Greater than or equal to y 理论 If the test result is positive, it is determined that the diquat formulation to be tested is not adulterated with paraquat.

[0029] In some embodiments, the expression for equation (1) is: y = 3512.6x + 1274.2;

[0030] The expression for equation (2) is: y = 0.9873e 0.072x ;

[0031] The expression for equation (3) is: y = -0.0046x + 0.9979.

[0032] In some embodiments, the mass ratio of the quaternary ammonium salt pesticide fluorescent probe to water is 1:2000 to 200000.

[0033] The principle of this disclosure is as follows:

[0034] This disclosure primarily involves preparing benzidine disulfonic acid derivatives from benzidine disulfonic acid, naphthalene anhydride, and triethylamine, using these derivatives as fluorescent probes for quaternary ammonium pesticides, and employing these probes for quantitative detection of quaternary ammonium pesticides. Benzidine disulfonic acid is a widely used chemical dye with advantages such as ease of preparation, good water solubility, and high electron density. Furthermore, its sulfonic acid groups can interact with cations, while the amino groups at both ends provide modification sites. Benzoic acid often acts as a catalyst in amidation reactions, and triethylamine is added as an acid-binding agent to the reaction between the anhydride and the amino group. Naphthalene anhydride is an important raw material for the synthesis of dyes, pigments, and fluorescent whitening agents. Forming a cyclic lactam with the amino groups at both ends of benzidine disulfonic acid provides a highly conjugated rigid structure, improving the insufficient fluorescence performance of benzidine disulfonic acid. Therefore, the quaternary ammonium salt pesticide fluorescent probe prepared in this disclosure possesses active groups such as carbonyl and sulfonic acid groups, enabling it to interact with quaternary ammonium salt pesticides through electrostatic and hydrogen bonding interactions. Through the combined effects of internal filtration and photoinduced electron transfer, the fluorescence emitted by the quaternary ammonium salt pesticide fluorescent probe under 305 nm excitation light is quenched at 480 nm. This achieves highly sensitive detection of quaternary ammonium salt pesticides using benzidine disulfonic acid derivatives as a fluorescent sensor. Furthermore, combined with the intrinsic fluorescence emission of diquat (340 nm), the fluorescence intensity at 340 nm increases with increasing diquat concentration, and the fluorescence intensity at 480 nm... The fluorescence intensity decreases as the concentration of paraquat increases. The fluorescence intensity at both 340 nm and 480 nm decreases, indicating that paraquat has a relatively small impact on the ratio of fluorescence intensity at 340 nm to 480 nm. Since this ratio has a linear relationship with the concentration of diquat, a mathematical model can be constructed to calculate the concentrations of the two pesticides using the fluorescence intensity at 340 nm and 480 nm. Specifically, the ratio of fluorescence intensity at 340 nm to 480 nm is used to calculate the diquat concentration, and then the paraquat concentration is calculated. This ultimately enables the identification and separate quantitative detection of diquat and paraquat, thus solving the problem of detecting paraquat adulteration in diquat.

[0035] The beneficial effects of this disclosure are as follows:

[0036] This disclosure uses common small organic molecules as raw materials to synthesize benzidine disulfonic acid derivatives through amidation reaction. The synthesis process is simple, the raw materials are readily available and low in cost, the synthesized benzidine disulfonic acid derivatives have high purity, good dispersibility in aqueous solution, and are easy to store, making them suitable for mass production.

[0037] Furthermore, the prepared benzidine disulfonic acid derivative was used as a fluorescent probe to achieve highly sensitive detection of quaternary ammonium pesticides. This method primarily relies on the electrostatic interaction between quaternary ammonium pesticides and the carbonyl and sulfonic acid groups on the surface of the benzidine disulfonic acid derivative, leading to fluorescence quenching of the fluorescent sensor. This sensor exhibits high sensitivity for the quaternary ammonium pesticide diquat, with a detection limit as low as 0.003 mg / L, and demonstrates good linearity within the range of 0.011 mg / L–2 mg / L. This method shows broad application prospects in pesticide residue detection and environmental pollutant detection. Attached Figure Description

[0038] Figure 1 The 1H NMR spectrum of the quaternary ammonium salt pesticide fluorescent probe prepared in Example 1 of this disclosure proves the correctness of the synthesized product.

[0039] Figure 2 The image shows the carbon NMR spectrum of the quaternary ammonium salt pesticide fluorescent probe prepared in Example 1 of this disclosure, which proves the correctness of the synthesized product.

[0040] Figure 3 This is the infrared spectrum of the quaternary ammonium salt pesticide fluorescent probe prepared in Example 1 of this disclosure. Figure 3 The absorption peak at the middle indicates that the formation of the amide bond causes benzidine disulfonic acid to covalently bind with 1,8-naphthoic anhydride.

[0041] Figure 4 The working curve of the prepared quaternary ammonium salt pesticide fluorescent probe for detecting diquat in Example 2 of this disclosure is used to illustrate the high detection sensitivity and good response.

[0042] Figure 5 The first working curve for detecting diquat formulation using the prepared quaternary ammonium salt pesticide fluorescent probe in Example 3 of this disclosure is used to illustrate the high detection accuracy.

[0043] Figure 6 The second working curve for detecting diquat formulation using the prepared quaternary ammonium salt pesticide fluorescent probe in Example 3 of this disclosure is used to illustrate the high detection accuracy.

[0044] Figure 7 The third working curve for detecting paraquat technical material using the prepared quaternary ammonium salt pesticide fluorescent probe in Example 3 of this disclosure is used to illustrate the high detection accuracy.

[0045] Figure 8 This is a schematic diagram illustrating the process of using the prepared quaternary ammonium salt pesticide fluorescent probe to detect the concentrations of diquat and paraquat in a sample, as described in Example 3 of this disclosure. Detailed Implementation

[0046] The present application will now be described in further detail with reference to the accompanying drawings and specific embodiments. Similar elements in different embodiments are referred to by related similar element reference numerals. In the following embodiments, many details are described to facilitate a better understanding of the present application. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other elements, materials, or methods. In some cases, certain operations related to the present application are not shown or described in the specification. This is to avoid obscuring the core parts of the present application with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.

[0047] Furthermore, the features, operations, or characteristics described in the specification can be combined in any suitable manner to form various embodiments. At the same time, the steps or actions in the method description can be rearranged or adjusted in a manner obvious to those skilled in the art. Therefore, the various orders in the specification and drawings are only for the clear description of a particular embodiment and do not imply a necessary order, unless otherwise stated that a particular order must be followed.

[0048] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this application.

[0049] The technical solution of this application is not limited to the specific embodiments listed below, but should also include various applications of using quaternary ammonium pesticide fluorescent probes for the identification of quaternary ammonium pesticides.

[0050] Example 1:

[0051] This embodiment describes a method for preparing fluorescent probes for quaternary ammonium salt pesticides, specifically following these steps:

[0052] 0.344 g (1 mmol) of benzidine disulfonic acid, 2 mL (14.4 mmol) of triethylamine, and 10 mL (96.1 mmol) of m-cresol were added to a three-necked flask, and the atmosphere was purged with nitrogen. The flask was heated in an oil bath for 4 hours at 80 °C. After heating, 0.594 g (3 mmol) of naphthalene anhydride and 0.488 g (4 mmol) of benzoic acid (as a catalyst) were added to the flask. The mixture was then heated in an oil bath with nitrogen continuously supplied for 18 hours at 180 °C. After the reaction was complete, the reaction system was cooled to room temperature, and 25 mL of ethyl acetate was added to allow the product to precipitate completely. After filtration, a white solid was obtained. The white solid was washed with 15 mL of methanol and 25 mL of ethyl acetate, and then dried thoroughly in a vacuum drying oven at 60 °C. The resulting white powder was a pure quaternary ammonium salt pesticide fluorescent probe, specifically a benzidine disulfonic acid derivative with the chemical formula C. 48 H 50 N4O 10 S2, its structural formula is:

[0053]

[0054] Figures 1-3 The results represent the characterization of the quaternary ammonium salt pesticide fluorescent probes prepared in this embodiment. Among them, Figure 1 The hydrogen NMR spectrum, after analysis, showed that the number and types of hydrogens were the same as those in the predicted structure, and a one-to-one correspondence between the spectrum and the hydrogens in the structure was established. The spectrum was also labeled, confirming the successful synthesis of the compound. Figure 2 The image is a carbon NMR spectrum. After analysis, δ164.17 corresponds to the carbonyl carbon, δ145.95 corresponds to the carbon bonded to the sulfonic acid, δ46.12 corresponds to the secondary carbon of the triethylamine salt, δ9.02 corresponds to the primary carbon of the triethylamine salt, and δ138.27-123.18 corresponds to the remaining 11 carbons, which confirms the successful synthesis of the compound. Figure 3 It's an infrared absorption spectrum, showing a value at 2936 cm⁻¹. -1 2976cm -1 2736cm -1 2677cm -1 CH stretching vibration from triethylamine salt, 2492 cm⁻¹ -1 The characteristic peak of triethylammonium salt is the NH stretching vibration at 3061 cm⁻¹. -1 3008cm -1 CH stretching vibrations on naphthalene and benzene rings, 1625 cm⁻¹ -1 1589cm -1 1469cm -1 1436cm -1 For the vibration of the aromatic skeleton, 779cm -1Absorption by out-of-plane bending vibration of naphthalene ring CH, 867 cm -1 846cm -1 700cm -1 The characteristic absorption peak of 1,2,4-trisubstituted benzene ring is 1708 cm⁻¹. -1 1666cm -1 It is a carbonyl stretching vibration, 1375 cm⁻¹ -1 The absorption peak for the CN stretching vibration of the amide bond is 1354 cm⁻¹. -1 The absorption peak for Ar-N stretching vibrations is 1238 cm⁻¹. -1 1190cm -1 The S=O peak represents an asymmetric stretching absorption peak at 1037 cm⁻¹. -1 The absorption peak is the S=O symmetric stretching vibration peak, 626 cm⁻¹. -1 This is the absorption peak of the CS stretching vibration.

[0055] Example 2:

[0056] This embodiment describes a method for detecting the residue concentration of the quaternary ammonium pesticide diquat using biphenyl disulfonic acid derivatives. The method is specifically implemented according to the following steps:

[0057] Step 1: Detection of diquat concentration in the standards: Water was used as solvent A. The quaternary ammonium pesticide fluorescent probe prepared in Example 1 was mixed with solvent A at a mass ratio of 1:10000 to obtain an aqueous solution of the quaternary ammonium pesticide fluorescent probe. 100 μL of the aqueous solution of the quaternary ammonium pesticide fluorescent probe was mixed with 900 μL of diquat aqueous solutions of different concentrations to obtain various standard solutions. The aqueous solution of the quaternary ammonium pesticide fluorescent probe without diquat was used as the blank group, and the aqueous solution of the quaternary ammonium pesticide fluorescent probe with diquat was used as the experimental group. The fluorescence spectra of all standard solutions and the fluorescence intensities at 340 nm (corresponding to the fluorescence emission of diquat) and 480 nm (corresponding to the fluorescence emission of the quaternary ammonium pesticide fluorescent probe) were recorded at an excitation wavelength of 305 nm. The fluorescence intensities of the blank group at 340 nm and 480 nm were recorded as F0 and F1, respectively, and the fluorescence intensities of the experimental group at 340 nm and 480 nm were recorded as F2 and F3, respectively. Based on the results obtained from the interaction between different concentrations of diquat and quaternary ammonium salt pesticide fluorescent probes, a graph was constructed showing the variation of y = (F2-F0) / (F3 / F1) (the ratio of fluorescence intensity) with the diquat concentration x. The working curve for detecting diquat concentration was obtained based on the fitting results. (See [link to graph]). Figure 4 The equation for the working curve is: y = 3614.1x + 4.048, with a linear range of 0.011 mg / L to 2 mg / L. The linearity R of the working curve is... 2 =0.9996, greater than 0.99.

[0058] Step 2: Detection of diquat residue concentration in actual rice samples: Take 2g of rice (rice can also be replaced with soybeans or urine), add 1mL of methanol and 1mL of water for extraction, then dilute the obtained extract 5 times with water, take 900μL and add 100μL of the quaternary ammonium salt pesticide fluorescent probe aqueous solution prepared according to Step 1 of this embodiment to obtain the sample. Record the fluorescence spectrum of the sample and the fluorescence intensity at 340nm and 480nm at an excitation wavelength of 305nm. Record the fluorescence intensity of the quaternary ammonium salt pesticide fluorescent probe aqueous solution without the sample at 340nm and 480nm as F0 and F1, respectively, and the fluorescence intensity of the quaternary ammonium salt pesticide fluorescent probe aqueous solution with the sample at 340nm and 480nm as F2 and F3, respectively. Substitute y=(F2-F0) / (F3 / F1) into the working curve obtained in Step 1 above to obtain the concentration of diquat in the sample.

[0059] Example 3:

[0060] This embodiment describes a method for detecting diquat and paraquat in commercial formulations of quaternary ammonium pesticides using fluorescent probes. Specifically, it describes a method for determining whether commercial diquat formulations are adulterated with paraquat and its concentration, which is completed according to the following steps:

[0061] Step 1: Determine the working curves for measuring the concentration of diquat and paraquat, respectively. There are two working curves for measuring the concentration of diquat, denoted as equation (1) and equation (2), respectively. The working curve for measuring the concentration of paraquat is denoted as equation (3). The determination steps are described as follows:

[0062] Determination of Equations (1) and (2): Take 900 μL of aqueous solutions of diquat technical grade (excluding paraquat) diluted with different concentrations, add 100 μL of the quaternary ammonium salt pesticide fluorescent probe aqueous solution prepared in step 1 of Example 2, and obtain various first standard solutions. The quaternary ammonium salt pesticide fluorescent probe aqueous solution without diquat technical grade dilution is used as the first blank group, and the quaternary ammonium salt pesticide fluorescent probe aqueous solution with diquat technical grade dilution is used as the first experimental group. Record the fluorescence spectra of all first standard solutions and the fluorescence intensities at 340 nm and 480 nm at an excitation wavelength of 305 nm. The fluorescence intensities at 340 nm and 480 nm of the first blank group are recorded as F0 and F1, respectively, and the fluorescence intensities at 340 nm and 480 nm of the first experimental group are recorded as F2 and F3, respectively. Based on the results obtained from the interaction between different concentrations of diquat and quaternary ammonium salt pesticide fluorescent probes, a graph was constructed showing the variation of y = (F2-F0) / (F3 / F1) (the ratio of fluorescence intensity) with diquat concentration x. The first working curve for detecting diquat concentration was obtained based on the fitting results, i.e., equation (1). (See [reference]). Figure 5The equation for the working curve is: y = 3512.6x + 1274.2, and the linearity R of the working curve is... 2 =0.9988, 0.13mg / L~40mg / L, greater than 0.99. Simultaneously, a graph was constructed showing the change of y=F3 / F1 (the ratio of fluorescence intensity) with the concentration x of diquat. Based on the fitting results, a second working curve for detecting the concentration of diquat was obtained, i.e., equation (2), see [reference]. Figure 6 The equation for the working curve is: y = 0.9873e 0.072x The linear range is 1 mg / L to 40 mg / L, and the linearity R of this working curve is... 2 =0.9980, greater than 0.99.

[0063] Determination of Equation (3): Take 900 μL of aqueous solutions of different concentrations of paraquat technical (assuming it does not contain diquat) after dilution, add 100 μL of the quaternary ammonium salt pesticide fluorescent probe aqueous solution prepared in step 1 of Example 2, and obtain various second standard solutions. The quaternary ammonium salt pesticide fluorescent probe aqueous solution without paraquat technical dilution is used as the second blank group, and the quaternary ammonium salt pesticide fluorescent probe aqueous solution with paraquat technical dilution is used as the second experimental group. Record the fluorescence spectrum and fluorescence intensity at 480 nm of all second standard solutions at an excitation wavelength of 305 nm. The fluorescence intensity at 480 nm of the second blank group is recorded as F1, and the fluorescence intensity at 480 nm of the second experimental group is recorded as F3. Based on the results obtained from the interaction between different concentrations of paraquat technical dilutions and quaternary ammonium pesticide fluorescent probes, a graph was constructed showing the change of y = F3 / F1 (the ratio of fluorescence intensity) with the concentration x of the paraquat technical dilution. The working curve for detecting paraquat concentration was obtained based on the fitting results, i.e., equation (3). (See [reference]). Figure 7 The equation for the working curve is: y = -0.0046x + 0.9979, with a linear range of 1 mg / L to 100 mg / L. The linearity R of the working curve is... 2 =0.9942, greater than 0.99.

[0064] Step 2, see Figure 8Detection of paraquat contamination in diquat samples: 900 μL of the diluted aqueous solution of the diquat formulation to be tested was taken as the sample, and 100 μL of the quaternary ammonium salt pesticide fluorescent probe aqueous solution prepared in step 1 of Example 2 was added to obtain the test solution. The quaternary ammonium salt pesticide fluorescent probe aqueous solution without the sample was used as the third blank group. The fluorescence spectra of the test solution and the third blank group and the fluorescence intensity at 340 nm and 480 nm were recorded at an excitation wavelength of 305 nm. The fluorescence intensities of the third blank group at 340 nm and 480 nm were recorded as F0 and F1, respectively, and the fluorescence intensities of the test solution at 340 nm and 480 nm were recorded as F2 and F3, respectively. Substituting y1=(F2-F0) / (F3 / F1) into equation (1): y=3512.6x+1274.2, the concentration x1 of diquat in the sample is calculated; then, substituting the concentration x1 of diquat in the sample into equation (2): y=0.9873e 0.072x In the above, the obtained y value is denoted as y. 理论 F3 represents the theoretical fluorescence quenching degree of the test solution without paraquat doping at 480 nm, which is also the fluorescence intensity of the test solution without paraquat doping at 480 nm. ’ The ratio of the fluorescence intensity F1 of the test solution at 480 nm to that of the third blank group (since the y value obtained by substituting x1 into equation (2) represents the ratio of the fluorescence intensity of the test solution containing x1 concentration of diquat at 480 nm to that of the third blank group at 480 nm, and the blank group was subsequently normalized, this ratio can be considered as the fluorescence intensity of the quaternary ammonium pesticide fluorescent probe aqueous solution at 480 nm in the presence of diquat), the ratio of the strong light intensity F3 of the test solution at 480 nm to the fluorescence intensity F1 of the third blank group at 480 nm is denoted as y. 实际 , that is, y 实际 =F3 / F1. Then through y 实际 With y 理论 The size of the fluorescence intensity at 480 nm of the test solution is used to determine whether it has been quenched by paraquat. 实际 Less than y 理论 If the fluorescence intensity of the test solution at 480 nm is quenched by paraquat, it is considered that the test diquat formulation is doped with paraquat. The equation Y = y is then used. 实际 / y 理论 Substituting into equation (3): y = -0.0046x + 0.9979, the final concentration of paraquat is obtained; if y 实际 Greater than or equal to y 理论 It was concluded that the fluorescence intensity of the test solution at 480 nm was not quenched by paraquat, meaning that paraquat was not added to the diquat solution.

[0065] The above description is only a preferred embodiment of this application. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. The application of a quaternary ammonium salt pesticide fluorescent probe in the fluorescence detection of diquat concentration, characterized in that, The quaternary ammonium salt pesticide fluorescent probe has the following structure: The application includes the following steps: The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and standard solutions containing different concentrations of diquat were added to obtain multiple standard solutions. The aqueous solution of the quaternary ammonium pesticide fluorescent probe without diquat was used as the blank group, and the aqueous solution of the quaternary ammonium pesticide fluorescent probe with diquat was used as the experimental group. Each standard solution was excited with fluorescence at 305 nm, and the fluorescence spectrum of all standard solutions and the fluorescence intensity at 340 nm and 480 nm were recorded. The fluorescence intensities of the blank group at 340 nm and 480 nm were recorded as F0 and F1, respectively, and the fluorescence intensities of the experimental group at 340 nm and 480 nm were recorded as F2 and F3, respectively. The working curve for detecting the diquat concentration was obtained by fitting the relationship between y=(F2-F0) / (F3 / F1) and the diquat concentration x. The quaternary ammonium salt pesticide fluorescent probe was dissolved in an aqueous solution, and an extract containing diquat was added to obtain a sample. The sample was subjected to fluorescence excitation at 305 nm. The fluorescence intensities F2 and F3 of the sample at 340 nm and 480 nm, and the fluorescence intensities F0 and F1 of the blank group at 340 nm and 480 nm were input into the working curve to obtain the concentration of diquat contained in the sample.

2. The application according to claim 1, characterized in that, The expression for the working curve is: y = 3614.1x + 4.

048.

3. The application of a quaternary ammonium salt pesticide fluorescent probe in the fluorescence detection of paraquat doped with diquat, characterized in that, The quaternary ammonium salt pesticide fluorescent probe has the following structure: The application includes the following steps: Step 1: Determine the working curves for measuring the concentration of diquat and paraquat, respectively. There are two working curves for measuring the concentration of diquat, denoted as equation (1) and equation (2), respectively. The working curve for measuring the concentration of paraquat is denoted as equation (3). The determination steps are described as follows: Determination of Equations (1) and (2): The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and aqueous solutions diluted with different concentrations of diquat technical were added to obtain multiple first standard solutions. The aqueous solution of the quaternary ammonium pesticide fluorescent probe without diquat technical dilution was used as the first blank group, and the aqueous solution of the quaternary ammonium pesticide fluorescent probe with diquat technical dilution was used as the first experimental group. Each first standard solution was subjected to fluorescence excitation at 305 nm, and the fluorescence spectra of all first standard solutions, as well as those at 340 nm and 480 nm, were recorded. The fluorescence intensity at 340nm and 480nm of the first blank group was recorded as F0 and F1, respectively, and the fluorescence intensity at 340nm and 480nm of the first experimental group was recorded as F2 and F3, respectively. The first working curve for detecting the concentration of diquat was obtained by fitting the relationship between y=(F2-F0) / (F3 / F1) and the concentration of diquat x, and was recorded as equation (1). At the same time, the second working curve for detecting the concentration of diquat was obtained by fitting the relationship between y=F3 / F1 and the concentration of diquat x, and was recorded as equation (2). Determination of Equation (3): The quaternary ammonium pesticide fluorescent probe was dissolved in an aqueous solution, and different concentrations of paraquat technical diluted in aqueous solution were added to obtain a variety of second standard solutions. The quaternary ammonium pesticide fluorescent probe aqueous solution without paraquat technical diluted solution was used as the second blank group, and the quaternary ammonium pesticide fluorescent probe aqueous solution with paraquat technical diluted solution was used as the second experimental group. Each second standard solution was subjected to fluorescence excitation at 305 nm, and the fluorescence spectrum and fluorescence intensity at 480 nm of all second standard solutions were recorded. The fluorescence intensity at 480 nm of the second blank group was recorded as F1, and the fluorescence intensity at 480 nm of the second experimental group was recorded as F3. The working curve for detecting paraquat concentration was obtained by fitting the relationship between y=F3 / F1 and the concentration x of paraquat technical diluted solution, which is recorded as Equation (3). Step 2, Detection of paraquat doping in diquat in the sample: Dissolve the quaternary ammonium pesticide fluorescent probe in an aqueous solution, add the diquat formulation to be tested to obtain the test solution, and use the aqueous solution of the quaternary ammonium pesticide fluorescent probe without the diquat formulation to be tested as the third blank group. Excite the test solution and the third blank group with fluorescence at 305 nm, and record the fluorescence intensities F2 and F3 of the test solution at 340 nm and 480 nm, and the fluorescence intensities F0 and F1 of the third blank group at 340 nm and 480 nm. Substitute y1=(F2-F0) / (F3 / F1) into equation (1) to obtain the concentration x1 of diquat in the test solution. Substitute x1 into equation (2) and record the obtained y value as y 理论 y 理论 F3 represents the fluorescence intensity of the test solution at 480 nm without paraquat doping. ’ The ratio of the fluorescence intensity F1 of the test solution at 480 nm to that of the third blank group is denoted as y. 实际 If y 实际 Less than y 理论 If so, it is determined that the diquat formulation to be tested is adulterated with paraquat, and Y=y 实际 / y 理论 Substituting into equation (3), we obtain the concentration of paraquat; if y 实际 Greater than or equal to y 理论 If the test result is positive, it is determined that the diquat formulation to be tested is not adulterated with paraquat.

4. The application according to claim 3, characterized in that, The expression for equation (1) is: y = 3512.6x + 1274.2; The expression for equation (2) is: y = 0.9873e -0.072x ; The expression for equation (3) is: y = -0.0046x + 0.9979.

5. The application according to any one of claims 1 to 4, characterized in that, The mass ratio of the quaternary ammonium salt pesticide fluorescent probe to water is 1:2000~200000.

6. The application according to any one of claims 1 to 4, characterized in that, The preparation of the quaternary ammonium salt pesticide fluorescent probe includes: Step 1: Dissolve benzidine disulfonic acid and triethylamine in a solvent and heat under inert gas protection to obtain a homogeneous solution A; Step 2: Add naphthalene anhydride and benzoic acid to solution A, and continue heating under the protection of the inert gas to obtain a homogeneous solution B; Step 3: Add ethyl acetate to precipitate the solute in solution B, then wash and dry the precipitated solute with detergent to obtain the quaternary ammonium salt pesticide fluorescent probe.

7. The application according to claim 6, characterized in that, The solvent is selected from m-cresol or p-cresol; The inert gas is selected from nitrogen, helium, or argon; The detergent is selected from any one or a mixture of methanol, ethanol, ethyl acetate and acetonitrile.

8. The application according to claim 6, characterized in that, The mass ratio of benzidine disulfonic acid, triethylamine and solvent is 1:1~5:5~25; The mass ratio of solution B, the naphthalene anhydride, and the benzoic acid is 1:0.2~1:0.3~1.

9. The application according to claim 6, characterized in that, The heating temperature in step one is 60℃~100℃, and the heating time is 0.5h~4h; The heating temperature in step two is between 120℃ and 200℃, and the heating time is between 12h and 48h.

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